The purpose of this project is to develop, on an expedited schedule, those inhibitors of anthrax lethal factor (LF) which are both effective and safe for use by humans. The US government has forcibly stated that an effective treatment for anthrax is a key national priority. In response, the PI, in close coordination with the key collaborators, has developed a logical, closely interrelated program, which will lead to mature, drug-like compounds. This modular program employs the most current and advanced structural, enzymological, NMR and in silico methodologies. The development will be conducted through essential, seamlessly interacting modules with well-defined interfaces between complementary functions. The integral modules are: sieving the known, commercially available chemical structures by virtual ligand screening (flexible LF protein-ligand docking) to identify the limited number of potential inhibitors, testing the potency of the selected structures in enzymological and NMR studies, selecting via the in vitro and cell tests the inhibitors with minimal cytotoxicity and effect on human matrix metalloproteinases (MMPs), performing co-crystallization and structural analyses at the angstrom level of the inhibitor-LF complexes and the subsequent refinement of the chemical structures to accomplish the best fit. Following two or three iterations involving each of the above modules, the team will re-evaluate the lead compound(s) and perform the required benchmarks prior to moving the drug candidate(s) into clinical trials. Our five specific aims are: (I) establish a comprehensive panel of purified human MMPs; (II) examine the substrate preferences of LF and identify the structure of its potential peptide substrates and protein targets; (III) design the most potent and selective inhibitors capable of efficiently blocking the cleavage function of LF; (IV) test the potency of the derivative library of LF inhibitors selected in Aim III; (V) examine cytotoxicity and inhibitory potency of optimized, fine-tuned compounds, and to identify the drug-like lead inhibitor(s) of LF for continued drug refinement and clinical trials. We are confident that the resulting LF antagonist(s) will efficiently and safely provide the therapy that is so urgently required.